逆转录病毒载体介导的标志基因转移研究

Retroviral-Mediated Reporter Gene Transfer in Human Leukemic Cells

目的：为建立高效、安全的逆转录病毒介导的基因标记系统。方法：用脂质体方法将含有标志基因 Ｎｅｏ Ｒ的逆转录病毒载体 Ｌ Ｘ Ｓ Ｎ 导入包装细胞 Ｐ Ａ３１７ ；通过与单向型包装细胞 Ｇ Ｐ Ｅ８６ 相互转导，获得高滴度的双嗜型产病毒细胞，并以其对人类白血病细胞进行基因标记。结果：脂质体转染法获得的病毒滴度约为２ ．０ ×１０５ Ｃ Ｆ Ｕ／ ｍｌ，而与 Ｇ Ｐ＋ Ｅ８６ 细胞转导后可提高至２ ．８ ×１０６ Ｃ Ｆ Ｕ／ ｍｌ；重组病毒转导白血病细胞（ Ｈ Ｌ－ ６０ ， Ｋ５６２ ， Ｋ Ｇ－ １） 后，聚合酶链反应（ Ｐ Ｃ Ｒ） 证实 Ｎｅｏ Ｒ 基因整合到靶细胞基因组，巢式 Ｐ Ｃ Ｒ 与补救分析均未能检测到辅助病毒。结论：“乒乓效应”可显著提高逆转录病毒滴度，而逆转录病毒介导的基因标记系统是安全的。

To develop an efficient gene marking system mediated by replicationincompetent retrovirus,the vector LXSN,containing Neo R gene,was transfected into amphotropic packaging cells PA317 by liposome transfection method.The retroviruscontaining supernatants were used to infect ecotropic packaging cells GP+E86. Then the PA317 cells were repeatedly infected by ecotropic virus from the GP+E86 producers to increase virus titer.This high titer retrovirus was applied to transduce the human leukemic cells.Results:The titer of liposome transfected PA317 pools assayed on NIH3T3 cells was 2.0×10 5 CFU/ml while the titer of GP+E86 cells transduced by amphotropic virus was 8.1×10 6 CFU/ml.By means of “ping-pong transduction”,the titer of PA317/Neo R cells could be markedly improved to 2.8×10 6 CFU/ml.Successful integration of Neo R gene into human leukemic cells (HL-60,K562,and KG-1) was verified by polymerase chain reaction(PCR).No helper virus could be found by both nested PCR and rescue assay.In conclusion,the so-called “ping-pong effect” could efficiently improve virus titer,and the safety of this gene transfer system might provide an optimal method for human gene marking/therapy.